Oligonucleotide which binds to a chromosomal binding site for p53 protein

ABSTRACT

The present invention provides methods for inhibiting cell growth by providing a growing cell with an oligonucleotide capable of binding to a chromosomal binding site for p53 protein. Moreover, in a preferred embodiment these methods can be used for preventing and treating cancer.

This is a continuation of application Ser. No. 07/879,618, filed on May1, 1992, now abandoned, which is a CIP application of U.S. Ser. No.02/863,661 filed on Apr. 6, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention relates to methods for inhibiting mammaliancellular replication. In a preferred embodiment, cell growth isinhibited in mammals for prevention and treatment of cancer. Accordingto the present invention, cell growth is inhibited by binding ofoligonucleotides to chromosomal sites normally bound by proteins, e.g.the p53 protein, whose normal function is to suppress uncontrolled celldivision.

BACKGROUND OF THE INVENTION

The process by which cellular replication occurs is complex, involvingmany steps and numerous factors including regulatory, cytoskeletal andpolymerization proteins. However, some early steps in this complexprocess are known to be essential, for example, initiation of DNAreplication.

In higher eukaryotes, DNA replication is thought to be initiated at manyorigins of replication present on chromosomal DNA. By virtue of homologyto known viral origins of replication, some highly repeated eukaryoticorigins of replication have been identified and sequenced (Jelinek etal. 1980 Proc. Natl. Acad. Sci. USA 77:1398-1402).

Moreover some proteins have been identified which have a role inregulation of cell growth. For example many of the cellularprotooncogenes are thought to have a normal role in cellular replicationwhich is improperly executed when the protooncogene becomes mutated. Thep53 gene is such a protooncogene. Normally the p53 protein appears toinhibit cell growth, however mutant p53 proteins can have an oppositeeffect upon cell growth, causing uncontrolled cell division and avariety of cancers, especially sarcomas, breast, brain, adrenal cortex,colon, lung and leukemic cancers (Finlay et al. 1989 Cell 57:1083-1093;Ben-David et al. 1991 Cell 66:831-834; and Haber et al. 1991 Cell64:5-8). Moreover, the p53 protein binds to DNA sites in asequence-specific manner (Kern et al. 1991a Science 252:1708-1711; Kernet al. 1991b Oncogene 6:131-136). However, the significance of DNAbinding by p53 protein relative to the role of p53 protein in cellularreplication has not been established.

According to the present invention, cell growth is inhibited bysite-specific oligonucleotide binding to DNA. Specifically, the site towhich the oligonucleotide binds is a DNA site which can be bound by aprotein repressor of cellular replication, e.g. the p53 protein.

Oligonucleotides have recently attracted attention as regulators ofnucleic acid biological function. Naturally occurring complementary, orantisense, RNA are used by some cells to control protein expression orplasmid replication. For example, replication of some Escherichia coliplasmids, including the ColE1 plasmid, is regulated by an antisense RNAcomplementary to an RNA primer of ColE1 DNA replication (Lacatena et al.1981 Nature 294:623-626; Lin-Chao et al. 1991 Cell 65:1233-1242).

Specific oligonucleotides have also been synthesized and tested asinhibitors of nucleic acid function. For example, splicing of a pre-mRNAtranscript essential for Herpes Simplex virus replication has beeninhibited with a linear oligonucleotide complementary to an acceptorsplice junction (Smith et al., 1986, Proc. Natl. Acad. Sci. USA83:2787-2791). A linear oligonucleotide has also been used to inhibitprotein synthesis of a human immunodeficiency virus (HIV) p24 protein(Agrawal et al. 1988 Proc. Natl. Acad. Sci. USA 85:7079-7083). Inanother example, linear oligonucleotides were used to inhibit HIVreplication in cultured cells. Linear oligonucleotides complementary tosites within or near the terminal repeats of the HIV retroviral genomeand within sites complementary to certain splice junctions were mosteffective in blocking viral replication (Goodchild et al., 1988, Proc.Natl. Acad. Sci. USA 85:5507-5511). Accordingly, the use ofoligonucleotides as inhibitors of nucleic acid function has been limitedto inhibition of functions such as RNA splicing, protein translation andviral replication via formation of Watson-Crick base pairs between anoligonucleotide and a nucleic acid template. The inhibition of cellgrowth by oligonucleotide binding has not been demonstrated.

An oligonucleotide binds to a target nucleic acid by forming hydrogenbonds between bases in the target and the oligonucleotide. Common B DNAhas conventional adenine-thymine (A-T) and guanine-cytosine (G-C) Watsonand Crick base pairs with two and three hydrogen bonds, respectively.The most common bonds that form between two complementary strands of RNAor DNA are Watson-Crick hydrogen bonds. However, other types of hydrogenbonding patterns are known wherein some atoms of a base which are notinvolved in Watson-Crick base pairing can form hydrogen bonds toanother, third, nucleotide. For example, thymine (T) can bind to an A-TWatson-Crick base pair via hydrogen bonds to the adenine, therebyforming a T-AT base triad. Hoogsteen (1959, Acta Crystallography 12:822)first described the alternate hydrogen bonds present in T-AT and C-GCbase triads. More recently, G-TA base triads, wherein guanine canhydrogen bond with a central thymine, have been observed (Griffin etal., 1989, Science 245:967-971).

Oligonucleotides have also been observed to bind and inhibit thefunction of a nucleic acid through non-Watson-Crick hydrogen bonding.For example, Cooney et al. (1988, Science 241:456) disclose a 27-basesingle-stranded oligonucleotide which bound to a double-stranded nucleicacid via non-Watson-Crick hydrogen bonds. This oligonucleotide inhibitedtranscription of the human c-myc gene in a cell free, in vitro assay bybinding to the c-myc promoter. In a review, Riordan et al. suggest thatlinear "switchback" oligonucleotides can be used to bind and inhibit thefunction of both strands of a double stranded nucleic acid target byWatson-Crick binding to one target strand and non-Watson-Crick bindingto the other target strand (Riordan et al. 1991 Nature 350:442-443.However, methods for inhibiting cell growth by either non-Watson-Crickor Watson-Crick binding of an oligonucleotide to a chromosomal bindingsite for a protein repressor of cellular replication are not availablein the prior art.

Accordingly, the present invention represents an innovative step forwardin the technology of cell cycle control by providing methods forinhibiting cellular replication through Watson-Crick andnon-Watson-Crick binding of an oligonucleotide to chromosomal sitesnormally bound by protein repressors of cellular replication.

SUMMARY OF THE INVENTION

The present invention is directed to a method for inhibiting mammaliancell growth by providing a growing mammalian cell with a cellgrowth-inhibiting amount of an oligonucleotide comprising an RNA or aDNA which binds to a chromosomal binding site for p53 protein andthereby inhibiting said mammalian cell growth.

An additional aspect of the present invention provides a method ofpreventing or treating cancer by administering to a patient atherapeutically effective amount of an oligonucleotide comprising an RNAor a DNA which binds to a chromosomal binding site for p53 protein.

Another aspect of the present invention provides methods for inhibitingin vitro mammalian cell growth by contacting cultured mammalian cellswith a cell growth inhibiting amount of an oligonucleotide comprising anRNA or a DNA which binds to a chromosomal binding site for p53 proteinand thereby inhibiting said in vitro mammalian cell growth.

A further aspect of this invention is directed to a method of inhibitingcell growth in a mammal or a cultured mammalian cell by binding one ofthe present oligonucleotides to a chromosomal site for p53 proteineither by Watson-Crick or non-Watson-Crick base pairs to form adouble-helical or a triple-helical oligonucleotide-DNA complex,respectively.

Yet another aspect of the present invention provides a composition whichincludes a therapeutically effective amount of an oligonucleotide whichcan bind to a chromosomal binding site for p53 protein and apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to inhibition of cellular growth byoligonucleotides capable of binding to chromosomal sites normally boundby protein repressors of cell replication. Moreover, the presentinvention contemplates inhibition of cellular replication in mammals forthe treatment and prevention of cancer as well as inhibition of culturedcell replication.

In particular, the present invention provides a method for inhibitingmammalian cell growth by providing growing cells with a cellgrowth-inhibiting amount of an oligonucleotide which can be an RNA or aDNA capable of binding to a chromosomal binding site for p53 protein,and thereby inhibiting mammalian cell growth. Moreover, according to thepresent invention, cell growth is preferably inhibited to treat orprevent cancer. In particular, this invention includes a method forpreventing or treating cancer by administering to a patient atherapeutically effective amount of an oligonucleotide which can be anRNA or a DNA which binds to a chromosomal binding site for p53 protein.

In another embodiment, the present invention provides a method forinhibiting in vitro mammalian cell growth by contacting culturedmammalian cells with a cell growth-inhibiting amount of anoligonucleotide which is an RNA or a DNA capable of binding to achromosomal binding site for p53 protein, and thereby inhibiting invitro mammalian cell growth.

A sequence for a chromosomal binding site for p53 protein is known (Kernet al. 1991 Science 252:1708-1711) and has been designated herein as aportion of SEQ ID NO: 1. The sequence of SEQ ID NO: 1, is depictedbelow:

                                 5'-TAAGCTTGATATTCTCCCCAGATGTAGTGAAAGCAGGTAGAT                                 TGCCTTGCC                                 3'-ATTCGAACTATAAGAGGGGTCTACATCACTTTCGTCCATCTA                                 ACGGAACGG                                 TGGACTTGCCTGGCCTTGCCTTTTCTTTCTTTCTTTCTTTCTTTA                                 TTACTTTCT                                 ACCTGAACGGACCGGAACGGAAAAGAAAGAAAGAAAGAAAGAAAT                                 AATGAAAGA                                 CTTTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT                                 CTTCTTCTT                                 GAAAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAA                                 GAAGAAGAA                                 CTTCTTCTTCTTTTTTTTTTGAGACAGAG-3'                                 GAAGAAGAAGAAAAAAAAAACTCTGTCTC-5'.

Portions of SEQ ID NO: 1 which can be chromosomal binding sites for p53protein include, but are not limited to, SEQ ID NO:2-5, SEQ ID NO: 7-9and SEQ ID NO: 13. SEQ ID NO: 2 and 3 correspond to positions 56-83 ofSEQ ID NO: 1, while SEQ ID NO: 4 and 5 correspond to positions 61-96 ofSEQ ID NO: 1. SEQ ID NO:7-9 correspond to positions 43-71, 70-95 and100-121 of SEQ ID NO: 1, respectively. SEQ ID NO: 13 corresponds topositions 60-83 of SEQ ID NO: 1. The sequences of SEQ ID NO: 2-5, 7-9and 13 are depicted below:

                                SEQ ID NO:2:                                        5'-CTTGCCTGGACTTGCCTGGCCTTGCCTTTTCTTTC                                        TTT;                                SEQ ID NO:3:                                        5'-AAAGAAAGAAAAGGCAAGGCCAGGCAAGTCCAGGC                                        AAG;                                SEQ ID NO:4:                                        5'-CTGGCCTTGCCTTTTCTTTCTTTCTTTCTTTCTTT                                        A;                                SEQ ID NO:5:                                        5'-TAAAGAAAGAAAGAAAGAAAGAAAAGGCAAGGCCA                                        G;                                SEQ ID NO:7:                                        5'-TGCCTTGCCTGGACTTGCCTGGCCTTGCC-3'                                        3'-ACGGAACGGACCTGAACGGACCGGAACGG-5';                                SEQ ID NO:8:                                        5'-TTTCTTTCTTTCTTTCTTTCTTTTCC;                                SEQ ID NO:9:                                        5'-CTTCTTCTTCTTTTTCTCTTTC; and                                SEQ ID NO:13                                        5'-TTTCTTTCTTTTCCGTTCCGGTCC-3'                                         3'-AAAGAAAGAAAAGGCAAGGCCAGG-5'.

According to the present invention a chromosomal binding site for p53protein is a domain within a chromosomal DNA having sufficient homologyto a nucleotide sequence corresponding to either or both strands of SEQID NO: 1, or a portion thereof, to permit detectable binding by p53protein. The size of chromosomal site for p53 protein can be about 10 toabout 200 bases or base pairs and is preferably about 10 to about 50bases or base pairs.

Binding of p53 protein to a chromosomal DNA can be detected by anyprocedure available in the art, for example by immunoprecipitation, gelshift, DNA footprinting, methylation interference and similar assays,for example, as provided in Finlay et al. (1989 Cell 57:1083-1093), Kernet al. (1991 Science 252:1708-1711) and Sambrook et al.(1989, MolecularCloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY).Moreover, recombinant p53 protein is available and can be obtained as apure or semi-pure preparation (Friedman et al. 1990 Proc. Natl. Acad.Sci. USA 87:9275; Hinds et al. 1989 J. Virol. 63:739-746; Finlay et al.1989 Cell 57:1083-1093). Furthermore, anti-p53 antibodies and kits fordetection of p53 protein can be obtained commercially (OncogeneSciences, Uniondale, N.Y.). Accordingly one of skill in the art canreadily utilize procedures and commercially available materials todetermine whether p53 protein binds to a DNA having a given sequence,and thereby ascertain the degree of homology to SEQ ID NO: 1 required bya chromosomal site to permit binding by p53 protein.

In a preferred embodiment, chromosomal DNA sites having greater thanabout 50%, and preferably greater than about 70%, homology to a portionof SEQ ID NO: 1 are sufficiently homologous to be chromosomal sitespermitting binding by p53 protein. Moreover, in an especially preferredembodiment, chromosomal sites are selected which can detectably bind atleast 50% or more of the p53 protein bound by a DNA having SEQ ID NO:1-4, SEQ ID NO: 7-9 or SEQ ID NO: 13.

According to the present invention a patient is a human. Moreover amammalian cell is an animal cell having a chromosomal DNA which encodesone or more copies of a binding site for p53 protein. Preferredmammalian cells are human, ape, monkey, mouse, rat, hamster, rabbit,cat, dog, horse, sheep, cow, bull and similar mammalian cells.Especially preferred mammalian cells of the present invention are human,monkey, mouse, rat and hamster cells.

Proliferation can be inhibited in numerous cells within a mammal, e.g.,to prevent or treat cancer, by the methods of the present invention. Inparticular, this invention has utility for inhibiting growth of any celltype which can have a mutant or absent p53 protein or which can becomecancerous when the p53 gene becomes mutated or is absent.

For example, according to the present invention, epithelial, mesothelialor endothelial cell growth can be inhibited in a mammal. Moreover, suchcell types can be cancerous or non-cancerous mesangial, embryonic,brain, lung, breast, uterine, cervical, ovarian, prostate, adrenalcortex, skin, blood, brain, bladder, gastrointestinal, colon and relatedcells. In a preferred embodiment cellular proliferation can be inhibitedin a mammal for treatment of a cancer. As used herein a cancer can be acarcinoma, sarcoma, breast, brain, adrenal cortex, colon, bladder,prostate, lung, leukemic or a related cancer.

According to the present invention, a mammalian cell having a mutant orabsent p53 protein, or a mutant or absent p53 gene, can be identified byany known procedure, including by immunological, sequencing orhybridization procedures. For example, cells expressing mutant p53protein can be distinguished from cells expressing wild type p53 proteinby commercially available antibodies which can distinguish between theseproteins (oncogene Science). Moreover, labeled nucleic acid probeshomologous to genomic DNA encoding p53 protein can be used to identifychanges in p53 mRNA or p53 DNA restriction fragment size by standardhybridization procedures (Sambrook et al.).

As used herein in vitro mammalian cellular proliferation means mammaliancell growth in culture. Moreover, mammalian cells whose proliferationcan be inhibited in vitro by the methods of the present inventioninclude any primary or immortalized cell line having a chromosomal DNAwhich encodes a binding site for p53 protein.

As used herein primary cell lines include cancerous and non-cancerouscells derived from any mammalian tissue specimen, for example, frommesangial,embryonic, brain, lung, breast, uterine, cervical, ovarian,prostate, adrenal cortex, skin, blood, brain, bladder, gastrointestinal,colon and related tissues. Immortalized cell lines can include humanHeLa, colon 201, neuroblastoma, retinoblastoma and KB cell lines, mouse3T3, L and MPC cell lines, hamster CHO and BHK 21 cell lines, a monkeyBSC cell line and other cell types, for example mammalian cell typesavailable from the American Type Culture Collection.

According to the present invention, cell growth is inhibited by bindingone or more of the subject oligonucleotides to a p53 protein chromosomalbinding site. Binding of the oligonucleotide has a similar effect uponcell growth as wild type p53 protein binding, i.e. inhibition of cellgrowth. Moreover, the methods of the present invention have been used toinhibit cell growth by up to 95%.

Without limiting the invention, binding of the present oligonucleotidesmay block access to that chromosomal binding site. Blocking access tothis site can prevent factors, e.g. nucleic acids or proteins, involvedin cellular or DNA replication from binding to or recognizing thechromosome at this site, from dissociating the two strands ofchromosomal DNA at this site, from moving along the chromosome, or fromrecognizing signals encoded within the chromosome at this site.Therefore, blocking access to the chromosomal binding site for p53protein may inhibit normal processes essential for cellular or DNAreplication and thereby may inhibit cell growth.

Inhibition of cell growth by the present oligonucleotides can beobserved in vitro or in vivo by any procedure available in the art. Forexample, in vitro inhibition can be detected by a reduction in number,or reduction in ³ H-thymidine uptake, of cultured cells treated with anoligonucleotide. Inhibition of cell growth in vivo, e.g., in a patientwith cancer, can be detected by any standard method for detecting tumorssuch as by X-ray or imaging analysis of a tumor size, or by observing areduction in mutant p53 protein production or in the production of anyknown cell-specific or tumor marker within a biopsy or tissue sample.

As used herein a cell growth-inhibiting amount of an oligonucleotide forinhibiting cell growth or proliferation in a mammal is about 0.1 μg toabout 100 mg per kg of body weight per day and preferably about 0.1 μgto about 10 mg per kg of body weight per day.

Moreover, a cell growth-inhibiting amount of an oligonucleotide forinhibiting in vitro cell proliferation or growth is about 0.1 μM toabout 100 μM of the oligonucleotide, and preferably about 1.0 μM toabout 50 μM of oligonucleotide.

In a preferred embodiment an oligonucleotide for inhibiting cellularreplication according to the methods of the present invention is adeoxyoligonucleotide having 10 to 100 bases. Moreover, oligonucleotideshaving any one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 12 are especiallypreferred oligonucleotides for inhibiting cell growth or for preventingand treating cancer.

However, according to the present invention any oligonucleotide havingsufficient complementarity to detectably bind to either strand of SEQ IDNO: 1, or a portion thereof, can be used to inhibit mammalian cellgrowth and to prevent or treat cancer.

Complementarity between nucleic acids is the degree to which the basesin one nucleic acid strand can hydrogen bond, or base pair, with thebases in a second nucleic acid strand. Hence, complementarity cansometimes be conveniently described by the percentage, i.e. proportion,of nucleotides which form base pairs between two strands or within aspecific region or domain of two strands. For the present inventionsufficient complementarity means that a sufficient number of base pairsexists between the subject oligonucleotides and a DNA having thesequence of either strand of SEQ ID NO: 1 to achieve detectable bindingof the oligonucleotide.

Therefore a sufficient number, but not necessarily all, nucleotides inthe present oligonucleotides can have hydrogen bonds to a DNA having thesequence of either strand of SEQ ID NO:1. The number of positions whichare necessary to provide sufficient complementarity for binding of thesubject oligonucleotides, can be detected by standard proceduresincluding known hybridization and melting temperature determinationprocedures. Moreover, according to the present invention oligonucleotidebinding can be detected by observing a decrease in cell proliferation,e.g., by measuring cell number or ³ H-thymidine uptake. Accordingly thedegree of complementarity between an oligonucleotide of the presentinvention and either strand of SEQ ID NO: 1 need not be 100% so long asoligonucleotide binding can be detected. However, it is preferred thatthe present oligonucleotides have at least about 50% complementaritywith either strand of SEQ ID NO: 1. In an especially preferredembodiment sufficient complementarity is greater than 70%complementarity with either strand of SEQ ID NO: 1.

Moreover, the degree of complementarity that provides detectable bindingbetween the subject oligonucleotides and SEQ ID NO: 1, is dependent uponthe conditions under which that binding occurs. It is well known thatbinding between nucleic acid strands depends on factors besides thedegree of mismatch between two sequences. Such factors include the GCcontent of the region, temperature, ionic strength, the presence offormamide and types of counter ions present. The effect that theseconditions have upon binding is known to one skilled in the art.Furthermore, conditions are frequently determined by the circumstancesof use. For example, the present oligonucleotides are used forinhibiting cell growth, accordingly no formamide will be present and theionic strength, types of counter ions, and temperature correspond tophysiological conditions. Therefore, oligonucleotides of the presentinvention are preferably selected by testing whether binding can occurto a DNA having SEQ ID No:1 under physiological salt and temperatureconditions when no formamide is present.

A thorough treatment of the qualitative and quantitative considerationsinvolved in establishing binding conditions that allow one skilled inthe art to design appropriate oligonucleotides for use under the desiredconditions is provided by Beltz et al., 1983, Methods Enzymol.100:266-285 and by Sambrook et al.(1989, Molecular Cloning: A LaboratoryManual, Vols. 1-3, Cold Spring Harbor Press, NY).

Thus for the present invention, one of ordinary skill in the art canreadily design a nucleotide sequence for the subject oligonucleotideswhich exhibits sufficient complementarity to detectably bind to achromosomal binding site for p53 protein. As used herein "binding" or"stable binding" means that a sufficient amount of the oligonucleotideis bound or hybridized to this site to permit detection of that binding.

Binding between a DNA encoding a chromosomal site for p53 protein and anoligonucleotide can be detected by any procedure known to one skilled inthe art, including both functional or physical binding assays.

Binding may be detected functionally by determining whether binding hasan observable effect upon cell growth, e.g., by observing a reducednumber of cultured cells or a reduced ³ H-thymidine uptake into culturedcells treated with the present oligonucleotides relative to untreatedcells.

Physical methods for detecting binding between complementary strands ofDNA or RNA are well known in the art, and include standard Southern andNorthern hybridization, light absorption detection, gel shift, DNAfootprinting, alkylation interference and related procedures (asprovided for example in Sambrook et al.).

According to the present invention oligonucleotides can bind to achromosomal site by conventional Watson-Crick base pairing or bynon-Watson-Crick base pairing.

Watson-Crick base pairing occurs through formation of base pairs betweenadenine (A) and thymine (T) or uracil (U) or suitable analogs thereof,and through formation of base pairs between guanine (G) and cytosine(C), or suitable analogs thereof. Moreover, Watson-Crick base pairingcan occur when oligonucleotide nucleotides are oriented in an opposite,or anti-parallel, 5' to 3' direction relative to nucleotides within achromosomal site. Binding of an oligonucleotide by Watson-Crick basepairing can be detected or assayed by any known hybridization procedure(Sambrook et al.)

In contrast to Watson-Crick base pairing, non-Watson-Crick hydrogenbonding occurs when oligonucleotide and chromosomal site nucleotideshave the same, or parallel, 5' to 3' orientation. Moreover, when thesubject oligonucleotides bind by non-Watson-Crick hydrogen bonding thestrands of the chromosomal site do not dissociate and a three strandedor triple-helical oligonucleotide-DNA complex forms. Accordingly, inanother embodiment the subject oligonucleotides bind by non-Watson-Crickbase pairing to form a triple-helical oligonucleotide-DNA complex, forexample as provided in Beal et al. (1991 Science 251:1360-1363), Mahleret al. (1990, Biochemistry 29:8820-8826), Haner et al. (1990Biochemistry 29:9761-9765), Maher et al. (1989, Science 245:725-730),Hoogsteen (1959, Acta Crystallography 12:822), Griffin et al. (1989,Science 245:967-971), Cooney et al. (1988 Science 241:456-459) or thelike.

In a preferred embodiment, non-Watson-Crick binding of the presentoligonucleotides occurs through formation of hydrogen bonds between oneof the present oligonucleotides and a double-stranded chromosomal DNAsite. In an especially preferred embodiment, non-Watson-Crick binding ofthe present oligonucleotides occurs through formation of hydrogen bondsbetween an oligonucleotide T and an A within an AT chromosomal site basepair, or between an oligonucleotide C and a G within a GC chromosomalsite base pair, to form T-AT and C-GC base triads, respectively.

Binding of an oligonucleotide by non-Watson-Crick hydrogen bonding toform a triple-helix oligonucleotide-DNA complex can be observed orassayed by art recognized procedures. Such procedures can includeoligonucleotide-directed affinity cleavage, DNase I footprinting,methylase interference and related procedures (Moser et al. 1987 Science238:645; Strobel et al. 1988 J. Am. Chem. Soc. 110:7927; Cooney et al.1988 Science 241:456-459).

Therefore, the skilled artisan can readily utilize the teachings of thepresent invention to make an oligonucleotide having the necessarystructural features to bind a chromosomal site with either Watson-Crickor non-Watson-Crick base pairs.

The present oligonucleotides are single-stranded DNA or RNA havingnucleotide bases guanine (G), adenine (A), thymine (T), cytosine (C) oruracil (U), or any nucleotide analog that is capable of hydrogen bondingby either Watson-Crick or non-Watson-Crick hydrogen bonds. Nucleotideanalogs include pseudocytidine, isopseudocytidine,3-aminophenyl-imidazole, 2'-O-methyl-adenosine, 7-deazadenosine,7-deazaguanosine, 4-acetylcytidine, 5-(carboxy-hydroxylmethyl)-uridine,2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thioridine,5-carboxymethylamino-methyluridine, dihydrouridine, 2'-O-methyluridine,2'-O-methyl-pseudouridine, beta,D-galactosylqueosine,2'-O-methylguanosine, inosine, N6-isopentenyladenosine,1-methyladenosine, 1-methyl-pseudouridine, 1-methylguanosine,1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine,2-methylguanosine, 3-methylcytidine, 5-methylcytidine, 5-methyluridine,N6-methyl-adenosine, 7-methylguanosine, 5-methylamino-methyluridine,5-methoxyaminomethyl-2-thiouridine, β-D-mannosylqueosine,5-methoxycarbonylmethyluridine, 5-methoxyuridine,2-methyl-thio-N6-isopentenyladenosine,N-(9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl)-carbamoyl)threonine,N-(9-beta-D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine. Whenpossible, either ribose or deoxyribose sugars can be used with theseanalogs. Nucleotide bases in an α-anomeric conformation can also be usedin the oligonucleotides of the present invention.

Preferred nucleotide analogs are unmodified G, A, T, C and Unucleotides; pyrimidine analogs with lower alkyl, lower alkoxy, loweralkylamine, phenyl or lower alkyl substituted phenyl groups in the 5position of the base and purine analogs with similar groups in the 7 or8 position of the base. Especially preferred nucleotide analogs are5-methylcytosine, 5-methyluracil, diaminopurine, and nucleotides with a2'-O-methylribose moiety in place of ribose or deoxyribose.

As used herein lower alkyl, lower alkoxy and lower alkylamine containfrom 1 to 6 carbon atoms and can be straight chain or branched. Thesegroups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tertiary butyl, amyl, hexyl and the like. A preferred alkyl group ismethyl.

The present oligonucleotides can be made by any of a myriad ofprocedures known for making DNA or RNA oligonucleotides. For example,such procedures include enzymatic synthesis and chemical synthesis.

Enzymatic methods of DNA oligonucleotide synthesis frequently employKlenow, T7, T4, Taq or E. coli DNA polymerases as described in Sambrooket al. Enzymatic methods of RNA oligonucleotide synthesis frequentlyemploy SP6, T3 or T7 RNA polymerase and reverse transcriptase asdescribed in Sambrook et al. To prepare oligonucleotides enzymaticallyrequires a template nucleic acid which can either be synthesizedchemically, or be obtained as mRNA, genomic DNA, cloned genomic DNA,cloned cDNA or other recombinant DNA. Some enzymatic methods of DNAoligonucleotide synthesis can require an additional primeroligonucleotide which can be synthesized chemically. Finally,oligonucleotides can be prepared by polymerase chain reaction (PCR)techniques as described, for example, by Saiki et al., 1988, Science239:487.

Chemical synthesis of oligonucleotides is well known in the art and canbe achieved by solution or solid phase techniques. Moreover,oligonucleotides of defined sequence can be purchased commercially orcan be made by any of several different synthetic procedures includingthe phosphoramidite, phosphite triester, H-phosphonate andphosphotriester methods, typically by automated synthesis methods.

Synthetic oligonucleotides may be purified by polyacrylamide gelelectrophoresis, or by any of a number of chromatographic methods,including gel chromatography and high pressure liquid chromatography. Toconfirm a nucleotide sequence, oligonucleotides may be subjected to DNAsequencing by any of the known procedures, including Maxam and Gilbertsequencing, Sanger sequencing, capillary electrophoresis sequencing thewandering spot sequencing procedure or by using selective chemicaldegradation of oligonucleotides bound to Hybond paper. Sequences ofshort oligonucleotides can also be analyzed by plasma desorption massspectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am.Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom.14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencingmethods are also known in the art for RNA oligonucleotides.

The present invention also contemplates derivatization or chemicalmodification of the subject oligonucleotides with chemical groups tofacilitate cellular uptake. For example, covalent linkage of acholesterol moiety to an oligonucleotide can improve cellular uptake by5- to 10-fold which in turn improves DNA binding by about 10-fold(Boutorin et al., 1989, FEBS Letters 254:129-132). Other ligands forcellular receptors may also have utility for improving cellular uptake,including, e.g. insulin, transferrin and others. Similarly,derivatization of oligonucleotides with poly-L-lysine can aidoligonucleotide uptake by cells (Schell, 1974, Biochem. Biophys. Acta340:323, and Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648).Certain protein carriers can also facilitate cellular uptake ofoligonucleotides, including, for example, serum albumin, nuclearproteins possessing signals for transport to the nucleus, and viral orbacterial proteins capable of cell membrane penetration. Therefore,protein carriers are useful when associated with or linked to theoligonucleotides of this invention.

Accordingly, the present invention contemplates derivatization of thesubject oligonucleotides with groups capable of facilitating cellularuptake, including hydrocarbons and non-polar groups, cholesterol,poly-L-lysine and proteins, as well as other aryl or steroid groups andpolycations having analogous beneficial effects, such as phenyl ornaphthyl groups, quinoline, anthracene or phenanthracene groups, fattyacids, fatty alcohols and sesquiterpenes, diterpenes and steroids.

In accordance with the present invention, modification in thephosphodiester backbone of oligonucleotides is also contemplated. Suchmodifications can aid uptake of the oligonucleotide by cells or canextend the biological half-life of such oligonucleotides. For example,oligonucleotides may penetrate the cell membrane more readily if thenegative charge on the internucleotide phosphate is eliminated. This canbe done by replacing the negatively charged phosphate oxygen with amethyl group, an amine or by changing the phosphodiester linkage into aphosphotriester linkage by addition of an alkyl group to the negativelycharged phosphate oxygen. Alternatively, one or more of the phosphateatoms which is part of the normal phosphodiester linkage can bereplaced. For example, NH--P, CH₂ --P or S--P linkages can be formed.Accordingly, the present invention contemplates usingmethylphosphonates, phosphorothioates, phosphorodithioates,phosphotriesters and phosphorus-boron (Sood et al., 1990, J. Am. Chem.Soc. 112:9000) linkages. The phosphodiester group can be replaced withsiloxane, carbonate, acetamidate or thioether groups. Thesemodifications can also increase the resistance of the subjectoligonucleotides to nucleases. Methods for synthesis of oligonucleotideswith modified phosphodiester linkages are reviewed by Uhlmann et al.(1990, Chemical Reviews 90:543-584).

A further aspect of this invention provides pharmaceutical compositionscontaining the subject oligonucleotides with a pharmaceuticallyacceptable carrier. In particular, the subject oligonucleotides areprovided in a therapeutically effective amount of about 0.1 μg to about100 mg per kg of body weight per day, and preferably of about 0.1 μg toabout 10 mg per kg of body weight per day, to bind to a nucleic acid inaccordance with the methods of this invention. Dosages can be readilydetermined by one of ordinary skill in the art and formulated into thesubject pharmaceutical compositions.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The subject oligonucleotides can be provided to a mammalian cell bytopical or parenteral administration, for example, by intraveneous,intramuscular, intraperitoneal, subcutaneous or intradermal route, orwhen suitably protected, the subject oligonucleotides can be orallyadministered. The subject oligonucleotides may be incorporated into acream, solution or suspension for topical administration. For oraladministration, oligonucleotides may be protected by enclosure in agelatin capsule. Oligonucleotides may be incorporated into liposomes orliposomes modified with polyethylene glycol for parenteraladministration. Incorporation of additional substances into theliposome, for example, antibodies reactive against membrane proteinsfound on specific target cells, can help target the oligonucleotides tospecific cell types.

Topical administration and parenteral administration in a liposomalcarrier is preferred.

The following examples further illustrate the invention.

EXAMPLE 1 INHIBITION OF CELLULAR REPLICATION BY OLIGONUCLEOTIDES

Oligonucleotides having the following sequences were synthesized byautomated DNA synthetic procedures:

                                           1                          p53(1)                              (SEQ ID NO:2):                                        5' CTTGCCTGGACTTGCCTGGCCTTGCCTTTTCTTTC                                        TTT;                          p53(2)                              (SEQ ID NO:3):                                        5'-AAAGAAAGAAAAGGCAAGGCCAGGCAAGTCCAGGC                                        AAG;                          p53(3)                              (SEQ ID NO:4):                                        5'-CTGGCCTTGCCTTTTCTTTCTTTCTTTCTTTCTTT                                        A;                          p53(4)                              (SEQ ID NO:5):                                        5'-TAAAGAAAGAAAGAAAGAAAGAAAAGGCAAGGCCA                                        G;                          p53(1R)                              (SEQ ID NO:6):                                        5'-CTTGCCTGGACGGTCCGTTCCTTGCCTTTTTTTCT                                        TTC;                          Hoog1                              (SEQ ID NO:8):                                        5'-TTTCTTTCTTTCTTTCTTTCTTTTCC;                          Hoog2                              (SEQ ID NO:9):                                        5'-CTTCTTCTTCTTTTTCTCTTTC;                          Hoog3                              (SEQ ID NO:10):                                        5'-CCCTTTTTTCCTTTTTTTCTTTTTCT;                          Hoog4                              (SEQ ID NO:11):                                        5'-TTTCTCTTTTCTTTTCTCCTTC;                          Hoog5                              (SEQ ID NO:12):                                        5'-TTTCTTTCTTTTCCTTTCCTTTCC.

Oligonucleotides having SEQ ID NO: 2 and SEQ ID NO: 4 are complementaryto oligonucleotides having SEQ ID NO: 3 and SEQ ID NO:5, respectively.Oligonucleotides having SEQ ID NO: 2 and 3 correspond to positions 56-83of SEQ ID NO:1 while oligonucleotides having SEQ ID NO: 4 and 5correspond to positions 61-96 of SEQ ID NO: 1. The oligonucleotidehaving SEQ ID NO: 6 (p53(1R)) is a control oligonucleotide having thesame size and base composition as SEQ ID NO: 2 but with two inversionsin nucleotide sequence between bases 11-20 and 31-38, relative to thenucleotide sequence of SEQ ID NO: 2.

Oligonucleotides having SEQ ID NO: 8 and SEQ ID NO: 9 correspond topositions 70-95 and 100-121 of SEQ ID NO: 1, respectively. Anoligonucleotide having SEQ ID NO: 12 has 87.5% homology to a region ofSEQ ID NO: 1 corresponding to positions 60-83. Oligonucleotides havingSEQ ID NO: 10 and 11 are controls having the same base composition andlength, but a different sequence, than SEQ ID NO: 8 and 9, respectively.

Oligonucleotides having SEQ ID NO: 2, 3, 4, 5, 8, 9 and 12 were made tobind in a parallel, i.e. non-Watson Crick, manner relative to the boundchromosomal DNA strand.

Oligonucleotides were synthesized on a Milligen 8750 DNA synthesizerusing phosphoramidite chemistry by LSUMC Core Laboratories, New Orleans.

Colon adenocarcinoma cells (Colo 201) and primary human mesangial cells(passage 5-6) were maintained in 80% RPMI (Gibco-BRL), 20% fetal bovineserum (FBS) and in 80% RPMI, 10% FBS with ITS supplement, respectively.ITS (Collaborative Research) contains 5 μg/ml insulin, 5 μg/mltransferrin and 5 ng/ml selenious acid. Each cell type was plated at7000 cells/microtiter well, grown for two days and then serum deprivedfor one day in media containing 0.5 % FBS. Low serum media were removedand replaced with media containing 10% FBS and 10 μM oligonucleotide orvehicle, i.e. a volume of water equivalent to the volume ofoligonucleotide solution added. Media and oligonucleotides were replacedevery 12 hr for a time period of 48 hr. During the final 24 hr, 10μCi/ml ³ H-thymidine (New England Nuclear), was added at each mediachange. Cells were harvested and bioassays performed using a TomtecHarvester 96 Mach II and an LKB 1205 Betaplate scintillation counter.Cells in parallel cultures were harvested, stained with trypan blue forviability and counted.

Table 1 depicts the effect of oligonucleotides having SEQ ID NO: 2-6upon cell growth of cultured colon and cultured mesangial cells asobserved by cell counting. When cultured colon carcinoma cells aretreated with an oligonucleotide having SEQ ID NO: 2 or SEQ ID NO: 4 cellcounts were inhibited by about 50%, relative to control cells receivingno oligonucleotide (vehicle, i.e. water). Similarly, oligonucleotidehaving SEQ ID NO: 2 inhibited cell counts of mesangial cells by about30%.

Under similar culture conditions, oligonucleotide SEQ ID NO:3(complementary to SEQ ID NO: 2) reduced colon and mesangial cell countsby about 30% and 11% respectively. However, an oligonucleotide havingSEQ ID NO: 5 with a complementary sequence to SEQ ID NO: 4 and acapacity to bind to SEQ ID NO: 1, had little effect upon colon cellcounts.

Table 2 depicts ³ H-thymidine incorporation into the DNA of cellscultured with an oligonucleotide having SEQ ID NO: 2-6 or without anoligonucleotide (i.e. vehicle). Oligonucleotides having SEQ ID NO: 2,SEQ ID NO: 3 and SEQ ID NO: 4 caused an approximate 50% reduction in ³H-thymidine incorporation into colon cells, relative to colon cellsreceiving no oligonucleotide. In contrast, an oligonucleotide having SEQID NO:5 had little or no effect upon cell growth as measured by ³H-thymidine incorporation. Moreover, control oligonucleotide SEQ ID NO:6, having the same base composition and a partially inverted sequencerelative to SEQ ID NO: 2 reduced ³ H-thymidine incorporation into coloncells by only 17%.

Growth of mesangial cells was less affected than was growth of coloncells by oligonucleotides having SEQ ID NO: 2 and 3. However, inhibitionof mesangial cell growth by these oligonucleotides was still significantas measured by ³ H-thymidine incorporation (Table 2). In particular,oligonucleotide SEQ ID NO: 2 caused a 22% reduction, and oligonucleotideSEQ ID NO: 3 caused a 13% reduction, in ³ H-thymidine incorporation intomesangial cells.

Oligonucleotides having SEQ ID NO: 8, 9 and 12 caused even greaterinhibition of cell growth as measured by ³ H-thymidine incorporationinto colon cells (Table 3). In particular, oligonucleotides with SEQ IDNO: 9 and 12 caused at least an 85% reduction in cell growth and anoligonucleotide with SEQ ID NO: 8 caused at least a 75% reduction incell growth as measured by ³ H-thymidine uptake. Controloligonucleotides having SEQ ID NO:10 and 11 caused no reduction in cellgrowth, and may have even increased cell growth slightly, indicatingthat the specific sequence of the oligonucleotide is a critical factoreffecting cell growth.

                  TABLE 1    ______________________________________    Cell Counts: Colon Carcinoma Cells and Human Mesangial Cells    Cultured with Oligonucleotides    ______________________________________    Colon Carcinoma Cells           SEQ ID NO:2    SEQ ID NO:3                                     Vehicle    ______________________________________           9,200          13,000     20,100           10,300         11,200     17,700           9,700          14,800     17,500    Mean   9,733          13,000     18,433    ______________________________________    Colon Carcinoma Cells          SEQ ID NO:4                     SEQ ID NO:5                                SEQ ID NO:6                                         Vehicle    ______________________________________          11,100     17,000     21,000   23,500          12,600     16,500     22,000   23,800          10,300     20,000     17,300   21,600    Mean  11,333.3   17,833.3   20,100   22,966.6    ______________________________________    Human Mesangial Cells           SEQ ID NO:2    SEQ ID NO:3                                     Vehicle    ______________________________________           20,000         22,500     26,000           15,000         20,150     22,000           16,000         21,600     24,200    Mean   17,000         21,417     24,067    ______________________________________

                  TABLE 2    ______________________________________    .sup.3 -H-Thymidine Incorporation (CPM's) into    DNA of Cells Cultured with Oligonucleotides    ______________________________________    Human Colon Carcinoma Cells           SEQ ID NO:2    SEQ ID NO:3                                     Vehicle    ______________________________________           10,884.7       11,908.8   20,109.1           11,654.2       12,346.8   19,789.0           10,111.8       11,994.7   21,008.4           11,039.6       11,853.6   18,435.1           8,408.1        11,198.6   18,095.6           9,882.7        9,497.2    19,850.7    Mean   10,330.2*      11,466.6*  19,548    ______________________________________    *p < 0.01, compared to vehicle (Wilcoxan Rank Sum)    Human Colon Carcinoma Cells           SEQ ID NO:2    SEQ ID NO:6.sup.†                                     Vehicle    ______________________________________           8,641.3        14,361.2   17,600.9           8,996.4        14,111.2   18,100.6           10,002.3       15,326.4   17,325.2           9,959.2        15,980.2   18,315.2           10,054.3       14,960.1   19,075.2           10,157.2       15,498.4    Mean   9,635.1        15,039.6*  18,083.4*    ______________________________________    *Different from SEQ ID NO:2 (p < 0.01) by Wilcoxan Rank Sum Test    .sup.† a partially inverted SEQ ID NO:2 sequence    Human Colon Carcinoma Cells          SEQ ID NO:4                     SEQ ID NO:5                                SEQ ID NO:6                                         VEHICLE    ______________________________________          12,105.2   20,417.0   16,012.8 24,009.5          11,824.3   20,277.9   10,064.9 19,156.8          8,217.4    13,907.1   23,793.3 22,165.2    Mean  10,715.6   18,200.6   19,957.0 21,777.2    ______________________________________    Cultured Mesangial Cells           SEQ ID NO:2    SEQ ID NO:3                                     Vehicle    ______________________________________           19,152.6       21,664.2   25,612.8           18,666.4       22,000.2   24,981.6           21,324.8       22,321.6   24,911.3    Mean   19,714.6       21,995.3   25,168.6    ______________________________________

                  TABLE 3    ______________________________________    Mean .sup.3 H-Thymidine Incorporation by Colo 201 Cells (in    ______________________________________    CPM's)    Vehicle 1        16,504.1     ± 2432    Vehicle 2        16,807.4    SEQ ID NO:8       4,205.0     ± 1840    SEQ ID NO:9       2,480.3     ± 686    SEQ ID NO:10     18,900.9     ± 2656    SEQ ID NO:11     19,152.7     ± 3492    SEQ ID NO:12      2,478.8     ± 210    ______________________________________

EXAMPLE 2 QUANTITATION OF MUTANT p53 PROTEIN IN CULTURED CELLS

The quantity of p53 protein in cultured colon carcinoma and humanmesangial was determined by an ELISA assay (Oncogene Sciences, New York)which can selectively detect mutant p53 proteins without significantlyreacting with wild type p53.

Materials and Methods

Cells were plated and grown to semi-confluence in T-175 tissue cultureflasks. Cell extracts were prepared using a non-ionic detergent methodto avoid denaturation of p53 proteins since denaturation can causeanti-mutant p53 antibodies to cross-reactive with wild type p53 protein.ELISA determinations were performed in parallel on cell extractsprepared from primary mesangial (MC) cell lines, which were expected tohave mostly wild type p53 protein, and from transformed colon carcinoma(Colo 201), cell lines, which were expected to have mostly mutant p53,using kits provided by Oncogene Sciences (New York). Extracts were alsomeasured for protein content using a Bio-Rad protein assay (Bio-RadLaboratories, Richmond, Calif.).

Results

Table 4 depicts the quantity of mutant p53 protein per unit volume andper mg of total cell protein. As shown, Colo 201 cells possessapproximately 12-fold more mutant p53 protein per mg of total cellprotein than do mesangial cells. The Colo 201 cell line was obtainedfrom a colon carcinoma. In contrast, the mesangial cells used in thisstudy are a primary cell line derived from normal, non-cancerous humankidney and have normal levels of wild type p53 protein. Furthermore, asshown in Example 1, oligonucleotides having SEQ ID NO: 2-5 inhibitcellular proliferation to a greater extent in Colo 201 cells than inmesangial cells. Therefore, even though there is substantially moreoncogenic mutant p53 protein in Colo 201 cells, cellular proliferationin Colo 201 cells is inhibited to a greater extent than is cellularproliferation in normal mesangial cells. Accordingly, the presentmethods can be used to selectively inhibit cellular proliferation incancerous cells.

                  TABLE 4    ______________________________________    Quantity of Mutant p53 Protein    in Cancerous and Non-Cancerous Cells                  Mutant p53 Mutant p53    Cell Line     (ng/ml extract)                             (ng/mg protein)    ______________________________________    Colo 201      52.5 + 6.5 6.60    Mesangial cells                   0.8 + 0.2 0.53    ______________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 13    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 188 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - TAAGCTTGAT ATTCTCCCCA GATGTAGTGA AAGCAGGTAG ATTGCCTTGC CT - #GGACTTGC      60    - CTGGCCTTGC CTTTTCTTTC TTTCTTTCTT TCTTTATTAC TTTCTCTTTT TC - #TTCTTCTT     120    - CTTCTTCTTC TTCTTCTTCT TCTTCTTCTT CTTCTTCTTC TTCTTCTTCT TT - #TTTTTTTG     180    #         188    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 38 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #     38           TGGC CTTGCCTTTT CTTTCTTT    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 38 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #     38           AGGC CAGGCAAGTC CAGGCAAG    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #       36         TTTC TTTCTTTCTT TCTTTA    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 36 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #       36         GAAA GAAAAGGCAA GGCCAG    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 38 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #     38           GTTC CTTGCCTTTT TTTCTTTC    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 29 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #            29    GCCT GGCCTTGCC    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #              26  TTTC TTTTCC    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #                 22CTT TC    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #              26  TTCT TTTTCT    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #                 22CCT TC    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    #                24TCCT TTCC    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #                24TCCG GTCC    __________________________________________________________________________

What is claimed:
 1. An oligonucleotide comprising SEQ ID NO: 8, SEQ IDNO: 9 or SEQ ID NO: 12.